This invention relates to microfabrication technology, such as DNA chip-making technology, and more specifically to methods and apparatuses for delivering controlled amounts of a solution to specific, closely spaced locations on a solid support.
In the fields of molecular biology and microbiology it has long been common in the art to make replicate arrays of biological agents to facilitate parallel testing of many samples. For example, the use of sterile velvet cloths and a piston-ring apparatus has long been used to make replicate agar plates of bacterial and yeast colonies on many plates, each containing a different growth medium, as a way of rapidly screening a large number of independent colonies for different growth phenotypes (Lederberg and Lederberg, J. Bacteriol. 63:399, 1952). Likewise, 96-well microtiter plates have long been used to store, in an organized and easily accessed fashion, large numbers of cell lines and virus isolates representing recombinant DNA libraries or monoclonal antibody cell lines.
Experimental screening of the 96-well microtiter plates housing a clone collection is commonly accomplished by using a rigid metal or plastic 96-pin device designed so that each pin is spaced relative to the others such that it fits precisely into the microtiter plate. Depending on the task at hand, the 96-pin device is lowered carefully to the surface of an nutrient-agar plate (if the objective was to grow replicate biological samples), into another microtiter plate (to grow or dilute the samples), onto nylon embranes (for molecular screening by DNA or RNA hybridization to identify a particular recombinant clone), or transferred for use in any other screening or procedure that is adaptable to the 96-well microtiter dish format.
While multiple prints may be performed from one pin dip into the samples arrayed in the master microtiter dish, the amount of sample deposited during each sequential print drops off. The ability to control the uptake of a solution onto the printing pin, and the deposition of solution onto a printing surface are critical to realizing an aliquotting devise which meets the technical needs of microarray production for the fields of genomics, molecular biology and molecular diagnostics.
An important factor in developing a successful printing process is the ability to control the force and speed of movement with which the pin tips contacts the surface being printing upon. As noted by Drmanac and Drmanac (BioTechniques 17:328, 335, 1994), two problems with conventional flat-cylinder pins are that drops can be caught on the sides of a pin leading to irregular printing, and drop splashing can occur when the printing pin head is withdrawn too fast from the printing surface. Too much force can lead to extensive damage to the print surface negating the utility of that print array. Too little force may be just as disabling in that variable amounts of sample may be transferred, or the print maybe defective all together. For example, when printing bacterial or viral samples to the surface of a nutrient-agar plate, too much pressure results in disruption of the agar surface, while too little force may result in little or no transfer of a sample. In addition, many nucleic acid hybridization membrane surfaces are fragile and are easily damaged by excess pin head force during sample printing.
The advent of large scale genomic projects and the increasing medical use of molecular diagnostics, has prompted the development of large volume throughput methods for screening recombinant DNA libraries representing entire genomes, the performance of large scale DNA sequencing projects, and executing replicative immunological assays, nucleic acid hybridization assays, or polymerase chain reaction assays. The following publications (and the references cited therein), which are exemplary only, provide general and specific overviews of large throughput methods that rely on biomolecular arrays, as well as methods of preparing such arrays: Eggers, M. D. et al. Advances in DNA Sequencing Technology SPIE Vol. 1891:113-126, 1993; Chetverin, A. B. et al. Bio/Technology 12:1093-1099, 1994; Southern, E. M. Nucleic Acids Research 22:1368-1373, 1994; Lipshutz, R. J. et al. BioTechniques 19:442-447, 1995; Schena, M. BioEssays 18:427-431, 1996; Blanchard, A. P. et al. Biosensors and Bioelectronics 11:687-690, 1996; O""Donnell-Maloney, M. J. et al. Genetic Analysis: Biomolecular Engineering 13:151-157, 1996; Regalado, A. Start-Up 24-30, October 1996; and Stipp, D. Fortune pp. 30-41, Mar. 31, 1997.
The need for high throughput methodology has led, in some cases, to a change from a 96-well microtiter dish format, to a 384-well (Maier et al., J. Biotechnology 35:191, 1994) or 864-well (Drmanac et al., Electrophoresis 13:120, 1992) format, which can also be used in conjunction with robotic devises (see, e.g., Belgrader et al., BioTechniques 19:426, 1995; Wilke et al., Diagnostic Microbiology and Infect. Disease 21:181, 1995). However, all of these automated techniques require the use of a robotic pin-tool devise that is capable of reproducibly transferring equal volumes of liquid from one arrayed configuration (i.e., 96-well microtiter plate) to another (i.e., 96-spot array on a hybridization filter membrane).
Recently, methods have also been developed to synthesize large arrays of short oligodeoxynucleotides (ODNs) bound to a glass surface that represent all, or a subset of all, possible nucleotide sequences (Maskos and Southern, Nucl. Acids Res. 20: 1675, 1992). Once such an ODN array has been made may be used to perform DNA sequencing by hybridization (Southern et al., Genomics 13:1008, 1992; Drmanac et al., Science 260:1649, 1993). The utility of this method of DNA sequencing would be greatly improved if better methods existed for the transfer and arraying of the precise amounts of the biochemical reagents required for the synthesis of large sets ODNs bound to hybridizable surfaces. This would enable greater equality of ODN yield at each position within the array and also increase the nucleotide chain length it is possible to synthesize.
The polymerase chain reaction (PCR) has found wide application to many different biological problems. Two major limitations to the commercial utilization of PCR are the high cost of the reagents and the inability to automate the performance of the process. Reagent costs can be lowered if the total volume of each reaction can be decreased, allowing a concomitant decrease in DNA polymerase and nucleotides. An accurate and reliable means to array small volumes of reagents using a robotically controlled pin tool could help solve both of these PCR problems.
As noted above, transfer devices have been in use for some time in the fields of microbiology and molecular biology. The types of devises which have been used can be roughly divided into two categories. Pressure devises (e.g., pumps and automatic pipettes), driven by positive and/or negative pressure, which deliver fixed aliquots of liquids sample via a pipette tip to a solid surface or into a microtiter well. Pipette arrays have been constructed that correspond to the standard 96-well microtiter dish format (Reek et al., BioTechniques 19:282, 1995). These devices are most accurate in the 5 xcexcl and above volume range, but are generally ill-suited to smaller volume tasks.
Solid surface pin devises transfer liquids based upon pin surface area and the factors regulating liquid surface tension, and have been widely adopted because of their simplicity and ability to transfer small volumes of liquid. These rigid pin devises have been used for several years in robotic devises to print multiple copies of nucleic acid microdot arrays which are then used in hybridization reactions to measure gene expression.
Researchers have modified the traditional rigid microarray printing tip so that it contains a micro-channel which functions by capillary action to collect and hold liquid for subsequent printing to a glass surface (Schena et al., Science 270:467, 1995; Schena, BioEssays 18.427, 1996; Shalon et al., Genome Res. 6:639, 1996). Such a print head has been used to print PCR amplified cDNA inserts into micro-arrays using a robotic system. Small volume (2 xcexcl per microdot) hybridization reactions were performed using this system to measure the differential expression of 45 genes by means of simultaneous, two color fluorescence hybridization (Schena et al., (Science 270:467, 1995).
There is a need in the art for highly efficient, cost effective means for arraying oligonucleotides and other biomolecules on a planar solid support. The present invention provides these and related advantages as disclosed in more detail herein.
In one aspect, the invention provides a spring probe comprising a tubular housing encasing a compression spring. The spring is in mechanical communication with a plunger. The plunger has a first region extending out of the housing, where the first region comprises a cone-shaped tip terminating in a flat surface. The flat surface is perpendicular to a longitudinal axis of the housing. The cone-shaped tip has, in cross-section, two exterior sides adjacent to the surface which, if the sides extended past the surface, would meet at a point positioned a distance of about 0.001-0.005 inches beyond the surface.
In another aspect, the invention provides a composition including a thickening agent at a concentration of about 35 vol % to about 80 vol % based on the total volume of the composition, an oligonucleotide at a concentration ranging from 0.001 xcexcg/mL to 10 xcexcg/mL, and water.
In another aspect, the invention provides a method for depositing a biomolecule onto a solid support. The method includes the steps of:
immersing a tip of a spring probe into a solution of biomolecule;
removing the tip from the solution to provide biomolecule solution adhered to the tip; and
contacting the biomolecule solution with a solid support to thereby transfer biomolecule solution from the tip to the solid support.
The spring probe used in the depositing includes a tubular housing encasing a compression spring, as described above.
In another aspect, the invention provides a method for arraying a biomolecule. The method includes the steps of:
immersing a tip of a spring probe into a solution of biomolecule;
removing the tip from the solution to provide biomolecule solution adhered to the tip;
contacting the biomolecule solution with a solid support to thereby transfer biomolecule solution from the tip to the solid support; and
repeating the contacting step a plurality of times to provide biomolecule patterned in an array on the solid support. Again, the spring probe having a tubular casing is as described above.
Other aspects of this invention will become apparent upon reference to the attached Figures and the following detailed description.